The present invention relates to a method for screening microbes antagonistic to pathogenic bacteria that emerge during raising of seedlings of gramineous plants by selection from a plant body, soil, a seed or seed soaking water utilizing tropolone resistance and tropolone non-producing characteristics as indices. Also, the present invention relates to a microbial pesticide containing as an active ingredients a bacterium and spores thereof characterized by having a resistance to tropolone and not producing tropolone and also having a antagonistic activity against pathogenic bacteria that emerge during raising of seedlings of gramineous plants.
Diseases that emerge during raising of seedlings of gramineous plants include rice xe2x80x9cBakanaexe2x80x9d disease, Helminthosporium leaf spot, and blast typically caused by fungi, rice bacterial gram rot, bacterial seedling blight, bacterial brown stripe, etc. caused by bacteria. To prevent these diseases, seed disinfectants, soil drenching agents, soil drench agents, foliage application agents after greening are said to be effective and these are used systematically by using them singly or incombination. For the disease damages by fungi, it has recently become possible to prevent them at high degree with EBI agents having high efficacy. Also, the disease damages caused by bacteria are prevented by use of chemical pesticides. However, chemical pesticides are generally low in their effect when bacterial diseases outburst and so that the problem remains that no sufficient prevention can be obtained. In case bacterial diseases outburst, at present the affected seedlings must be abolished. Therefore, development of a pesticide that can prevent or control the outburst of bacterial diseases has been desired.
Under the circumstances, the present inventors have made intensive investigation with view to making a research on bio-controlling material that is superior in controlling effect to chemical pesticides on the bacterial diseases of gramineous plants. Focusing on tropolone, which is a pathogenic toxin of pathogenic bacteria on rice bacterial seedling blight disease, they have searched a bacterium that is resistant to tropolone but does not produce tropolone from rice plants and established a method for screening a antagonistic bacteria to the pathogenic bacteria. Using this screening method, they have made an extensive search for microbial materials that can be used for controlling bacterial diseases. As a result, they have been successful in isolating antagonistic bacteria from rice seedlings that are antagonistic to the pathogenic bacteria.
The present invention relates to a method for screening a microbe antagonistic to pathogenic bacteria that emerge during raising of seedlings of gramineous plants by selecting a microbe from plant body, soil, seeds or seed soaking water utilizing tropolone resistance and tropolone non-producing property as indices. Also the present invention relates to a microbial pesticide containing as an active ingredient, the bacterium that is characterized by tropolone resistance and tropolone non-producing property selected by the screening method or spores thereof. Specifically, the present invention relates to a microbial pesticide comprising as a bacterium having a antagonistic action against pathogenic bacteria on rice plants, especially those that emerge during raising rice seedlings, a bacterium selected from the group consisting of bacteria belonging to the genera Pseudomonas, Bacillus, and Enterobacter. Also the present invention relates to a microbial pesticide comprising a mixture of two or more out of these. Specifically, the present invention relates to amicrobial pesticide for controlling bacterial diseases of gramineous plants, characterized in that one of bacteria belonging to either one of the genera Pseudomonas, Bacillus, and Enterobacter that have a antagonistic activity to pathogenic bacteria for bacterial diseases emerging in rice plants or a mixture of two or more of these is used for seed treatment or caused to be contained by soil for raising seedlings, or the bacterial cells are added to a seed soaking water, or the bacterium-containing solution is sprayed onto a rice plant body in the field.
The screening method of the present invention is carried out by placing or spreading a fragment of a plant such as rice plant, homogenate of plant, soil, a seed, and a seed soaking water, on a suitable solid medium containing tropolone transplanting the resulting colonies of a tropolone resistant bacterium on a suitable solid medium containing iron ion, and selecting a colony that shows no red color on the iron-containing medium, cultivating the bacterium on a suitable medium to grow it, collecting the bacterial cells, converting the cells to a formulation or directly treating the infected seeds with a live bacterium suspension, and then raising seedlings and examining the protective activity to select an effective strain. In the screening of the present invention, tropolone derivatives may also be used instead of tropolone.
The bacterium that can be selected by the screening of the present invention and has a antagonistic activity to the pathogenic bacteria emerging during raising seedlings of gramineous plants, or spores of the bacterium can be utilized as an active ingredient of a microbial pesticide. The bio-controlling material that are used in the present invention are obtained by the above screening method and include, for example, bacteria belonging to either one of the genera Pseudomonas, Bacillus, and Enterobacter or two or more of them may be used in admixture. The bacterium belonging to the genus Pseudomonas includes, for example, Pseudomonas aureofaciens, and more specifically Pseudomonas aureofaciens B5. The bacterium belonging to the genus Bacillus includes, for example, Bacillus polymyxa and more specifically Bacillus is Bacillus polymyxa B36. The bacterium belonging to the genus Enterobacter includes, for example, Enterobacter cloacae and more specifically Enterobacter cloacae B51. The bacteriological characteristics of Pseudomonas aureofaciens B5, Bacillus polymyxa B36 and Enterobacter cloacae B51 will be indicated below.
These three bacteria were isolated from rice seedlings by the present inventors. From the above characteristics, these were identified as Pseudomonas aureofasciens, Bacillus polymyxa and Enterobacter cloacae referring to Bergy""s Manual of Systematic Bacteriology Vol. 1, Vol. 2, and Bergy""s Manual of Systematic Bacteriology (Ninth Edition). The bacteria were named Pseudomonas aureofaciens B5, Bacillus polymyxa B36, and Enterobacter cloacae B51 respectively. The bacteria have been deposited at National Institute of Bioscience and Human Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry under FERM BP-6067, FERM BP-6068, and FERM BP-6069.
The bacteria are resistant to tropolone and are tropolone non-producing. They have a antagonistic activity to bacteria emerging in gramineous plants, mainly pathogenic bacteria causing bacterial disease damages. Examples of disease damages against which they are effective include rice bacterial seedling blight (Pseudomonas plantarii), rice bacterial gram rot (Pseudomonas glumae), rice bacterial brown stripe (Pseudomonas avenae), rice bacterial leaf blight (Xanthomonas campestris), rice bacterial halo blight (Pseudomonas syringae pv. Oryzae), rice bacterial foot rot (Erwinia chrysanthemi pv. Zeae), rice bacterial palea browning (Erwinia herbcola), rice bacterial sheath brown rot (Pseudomonas fuscovaginae), rice xe2x80x9cBakanaexe2x80x9d disease (Gibberella fujikuroi), rice Helminthosporium leaf spot (Cochliobolas miyabeanus), rice blast (Pyricularia oryzae), barley/wheat bacterial black node (Pseudomonas syringae pv. japonica), oat bacterial halo blight (Pseudomonas syringae pv. coronafaciens), oat bacterial stripe blight (Pseudomonas syringae pv. striafaciens), corn bacterial brown stripe (Pseudomonas avenae), corn bacterial stalk rot (Erwinia chrysanthemi pv. zeae), fescue/rye grass halo blight (Pseudomonas syryngae pv. atropurprea), sorghum bacterial leaf stripe (Pseudomonas andropogonis), Zoysia grass Rhizoctonia disease (Rhizoctonia solani), zoysia grass Rhizoctonia rot (Rhizoctonia) etc.
The microbial pesticide of the present invention comprises the bacteria and/or spores thereof as an active ingredient. Suspensions of the above bacteria are spread, sprayed, or coated on, for example, a gramineous plant or seeds thereof or gramineous plant seeds are dipped in the suspensions of the bacterial or the bacterial cells are added to seed soaking water, drenched or mixed in soil for raising seedlings (bed soil or cover soil), or the bacterial suspension is applied to the gramineous plant body in the field. Suitable concentrations of the bacteria may vary depending on the above methods of application. For example, in the case where rice seeds are subjected to dip treatment, it is conducted with the bacteria in a concentration of 1xc3x971011 to 1xc3x97102/ml, preferably 1xc3x971010 to 1xc3x97105/ml, at 5 to 40xc2x0 C., preferably 15 to 30xc2x0 C., for 1 to 2 days. In this case, the suspension may be a culture broth. Alternatively, the medium may be removed from the culture broth by, for example, centrifugation and the cells may be resuspended in water or physiological saline, buffer, etc. before use. Also, there may be used a solution obtained by resuspending cells that have been stored in a stable state by freeze-drying directly or with a suitable dispersant.
The microbial pesticide of the present invention may be one that is a suspension of the above antagonistic bacteria and/or spores thereof having a antagonistic activity to the pathogenic bacteria emerging during raising gramineous plant seedlings as they are. However, they may be mixed with various carriers such as a solid carrier or liquid carrier, and further with additives and other formulation aids, if necessary, and used as formulations prepared as wettable powder, suspension, powder, granule, paste, and microcapsule.
The solid carriers used upon formulation include, for example, mineral carriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid white soil, vermiculite, and pearlite, and inorganic salts such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride, and calcium carbonate. Also, organic fine powders such as wheat flour, wheat bran, and rice bran may be used. The liquid carriers include vegetable oils such as soybean oil and cottonseed oil, glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, etc.
The formulation of bonds and dispersants include polysaccharides such as casein, gelatin, starch, gum Arabic, alginic acid, and cellulose derivatives, lignin derivatives, saccharides, vegetable oils, mineral oils, synthetic water soluble polymers, etc.
Besides, as the formulation auxiliaries, there can be used anti-freezers, defoaming agents, thickening agents, etc., as needed.
The antagonistic bacteria of the present invention can be used in admixture with seed disinfectants or sprays for disease damages caused by fungi. For example, there can be cited ipconazol, pefurazoate, triflumizole, prochloraz, fludioxonil, benomyl, thiophanate-methyl, fthalide, tricyclazol, pyroquilon, carpropamid, ferimzone, isoprothiolane, EDDP, carboxine, etc. Further, the antagonistic bacteria of the present invention may be used with or without mixing with insecticides, nemotocides, acaricides, fungicides, herbicides, plant growth regulator, synergists, etc. simultaneously.
The formulations of the microbial pesticide of the present invention may contain usually about 0.1 to 95% by weight in a wet weight of the antagonistic bacteria of the present invention. It is preferred that the formulation contains about 103 to about 1010 colonies per g of formulation.